Cyclopentanopolyhydrophenanthrene derivatives



Patented Jan. 29, 1952 CYCLOPENTANOPOLYHYDROPHENAN- 'THRENE DERIVATIVESMaximilian R. Ehrenstein, Philadelphia, Pa., as-

signor to The Trustees of the University of Pennsylvania, Philadelphia,Pa., a corporation of Pennsylvania No Drawing. Application December 20,1950,

Serial No. 201,887

'7 Claims.

This invention is in the art of cyclopentanopolyhydrophenanthrenes andconcerns specifically a group of estranes in it, which arederiva tivesof, and include 3,5-dihydroxy-10-hydroxymethyl-l'7-carboxy-estrane, andwhich group embraces primarily three major types of derivatives, namely:(at) those in which there is no unsaturation between the carbon atoms inthe-8-, 14- and 15-positions; (b) those in which there is an oxygenlinkage between the 8- and lQ-carbon atoms; and those in which there isa double bond linkage between the carbon atoms in the 14- and15-positions. In addition to these three major types of derivatives,within each of the types are included also those derivatives in which(1) the hydroxyl groups and the 17-carboxyl group remain unchanged; and(2) those in which the hydroxyl groups remain unchanged but thel'l-carboxyl group is. esterified, particularly with an alkyl or anaralkyl alcohol, andthereby converted to a carbalkoxy or carbaralkoxygroup; and also those (3) in which in addition to the 1'?- carbalkoxy or-carbaralkoxy group, both the hydroxyl group linked to the 3-carbon atomand the one (if present) linked to the 19-carbon atom are esterifiedwith particularly a carboxylic acid (either aromatic, aralkyl, or afatty acid), whereby both are converted to an acyloxy group, al-- thoughthis esterification can also be with an aromatic sulfonic acid; as wellas those (4.) in

which the 5-hydroxy, and the lI-carboxy groups remain intact but the3-hydroxy group and the 19-hydroxy group (if present) are esterifiedun-- der ordinary mild esterification conditions for conversion toeither a carboxylic acid ester or, an aryl sulfonic acid ester group.

Accordingly, the products of the invention include (I) thel'l-carbalkoxy derivatives of the above thre difierent major types of3,5-dihydroxy-lO-hydroxy-methyl compounds, namely, of (a)3,5,19-trihydroxy-etiocholanic acid, and of (b) the one having thecarbon atoms in the 8- and 19-positions linked by oxygen with only asingle bond linking the carbon atoms in the 14- and 15-positions, aswell as (c) the other having the carbon atoms in the 8- and'l4-positions linked only by a single bond while those in the 14- and15-positions are linked by a double bond; and (II) the ll-freecarboxylic acid of each of the three types included under (I) and also(III) the l7-alkyl and -aralkyl esters of the 3,19-

dialkoyloxy-5-hydroxy-etiocho1anic acid; as well part of the preceding.paragraph, for example, by use of a carboxylic acid anhydride or an arylsulfonic acid.

The above described various types of compounds included in the groupembraced by the invention are obtained by a procedure commencing with3,5,14,l9-tetrahydroixy-l4-isoetiocholanic acid, the preparation ofwhich is described in detail in my application Serial No. 666,191, filedApril 30, 1946, now Patent No. 2,518,672.

In the procedure for obtaining the products of the invention, the3,5,14,1S-tetrahydroxy-l-isoetiocholanic acid is first subjected todehydration, for example, by the use of an alcohol, preferably anabsolute lower alkyl alcohol as ethyl alcohol, acidulated with mineralacid such as sulfuric acid or preferably hydrogen chloride, andpreferably under anhydrous conditions, whereby the hydroxyl group in thelei-position ofthe starting material is removed with resulting (a)formation of an oxygen linkage between the 8- and 19-carbon atoms or (b)unsaturation between the 14- and 15-carbon atoms, and largely withconcurrent esterification of the 1.7-carboxy group to a carbalkoxygroup. This dehydration (removal of the hydroxyl from the li-carbon)resulting in the indicated unsaturation or formation of anether bridge,and accompaniedby the simultaneous esterification of the l'l-carboxylgroup is a unique feature of this invention.

In the subsequent working up of the reaction products of the dehydrationstep, there are obtained neutral products to the extent of about andacid products to the extent of about 15%. From the neutral products bysubsequent teratment there are isolated the two alkyl etiocholenic oretiocholanic acid esters (the one with the oxido linkage between the 8-and 19-carbon atoms, and the other with ri -unsaturation). By subsequentcatalytic hydrogenation, using platinum (or palladium, rhodium, ornickel), the A ester is saturated to the corresponding alkyl3,5,19-trihydroxy-etiocholanate. By subsequent saponification, from eachof these types of esters, there is obtained the correspondingetiocholanic acid. V

Among the approximately 15% of acid prodnets of the dehydration, therecan be found the corresponding free A -etiocholenic acid.

Each of the above noted three free acids, (at) thesaturated'one; (b) theoxygen bridged one, and (c) the one that isunsaturated, can be esterified throughthe17-carboxyl group by reac tion with a dia'zo reagent,such'as adiazo alkane dles.

esterified at both the 3-hydroxyl group and the 19-hydroxyl group, forexample, with a fatty, aromatic or arylaliphatic acid, or with anaromatic sulfonic acid, or any of the types of free acids can besimilarly esterified at the same two hydroxyl groups and with the5-hydroxy1 and the 17-carboxyl groups remaining intact,

The invention is illustrated by, but not restricted to, the followingexamples:

Example J.-Ethyl 3,5,1 9-trihydromy-A etiocholenate 2.5088 grams ofcrystalline 3,5,14,19-tetrahydroxy-M-isoetiocholanic acid having thestructure:

i9 is onion CH: COOK V H was dissolvedin 240 cc. of a one-tenth normalsolution of hydrogen chloride in absolute alcohol.

The resulting solution was kept at room temperajture'for minutes,and'then subjected to a slow distillation begun at atmospheric pressureand under anhydrous conditions by using a paraffin bath which was keptat between 103 and 105 0. throughout the entire distillation which ranfor 95 minutes. To the distillation residue, 75-

80 cc. of a 'lightgolden solution, 50 cc. of Water was added and thedistillation then continued under vacuum (-47 C.) until a distinctturbidity appeared. The material causing the turbidity was brought backinto the solution by briefly heating the mixture on a water bath. Thesolution was then allowed to stand overnight at room temperature,resulting in the separation of some oily material and alsomacrocrystalline nee- Since the crystals were partly embedded in theoil, as much of the 'oil as possible was decanted from the mixturewithout taking over crystals, and adhering'oil was removedfrom the oiland crystal mixture by washing the mixture by decantation with severalportions of one to one aqueous alcohol until thecr-ystals were left intheir colorless form. All of these decantates were combined and thealcohol removed completely by distillation under vacuum. A light yellowprecipitate formed in the aqueous mixturein the distillation fiask andthe whole mixture was shaken up with 175 cc. of ether. The aqueous layerwas removed and then extracted four times with separate 75 cc. portionsof ether. These four ether extracts were combinedwith the original etherlayer, and

V the whole was-then washed six times, each with 5 cc. of water, andthendried with anhydrous sodiumjsulf'ate, and filtered. After removing theether under vacuum from the filtrate, the residue was a brittle'foam ofdry weight 2.3531. grams. This residue was then dividedintoapproximately two equal parts for convenience in further proc- 4essing. These are identified oily residue A and oily residue B.

Oily residue A was separated into its neutral and acid portions bydissolving 1.2273 grams of it in cc. of ether and extracting it. firstwith 10 cc. and then 5 cc. of a 5% solution of sodium carbonate and thenfive times with 2 cc. portions each of water, thereby removing the acidpor tion. The neutral part remained in the ether solution which was thendried with anhydrous sodium sulfate and filtered. The ether was thenremoved under vacuum and the dry residue, neutral portion, was acolorless brittle foam weighing 1.0 177 grams.

This dry residue, neutral portion, was dissolved in 5 cc. of ether to aclear, yellowish solution, to which some'petroleum ether was added adrop or two at a time, with shaking, over the course of 8 hours, duringwhich very gradually some hard, white crystalline material deposited onthe walls of the flask. The amountzincreased on standing over night. Thefollowing morning the crystals were filtered off and washed with ethercontaining petroleum ether, and then worked .up

to isolate their specific product content'as' described in ExampleXVIIKcolumn 1'7). 7 i

To the mother liquor (filtrate from these crystals) was added petroleumether, in the same way as above over a period of several hours,resulting in the precipitation of only resinous material. This was notseparated but rather brought to dryness together with the supernatantsolution to give as a dry;residue,0.9473 gram of a brittle foamcontaining more-than one substance, but mainly crude ethyl3,5,19-trihydroxy-a etiocholenate, showing optical rotation Mig e -46.4

- (20.0 mg. in 2.0 cc. or chloroform). The chloroform solution of thesubstance gave a yellow 'color with tetranitromethane, indicating theunsaturated character. In a repeat run, the resinous material,precipitated on the addition of petroleum ether, was separated and takenup in acetone, from which it crystallized in flat prisms.

Still another run was carried through the dehydration to the point ofobtaining the oily residue A. After removing from the neutral portion ofthe oily residue A the crystalline substance contained in it (asmentioned in the s'ec- 0nd paragraph above), the resulting filtrate wasbrought to dryness. This dry residue was taken up in acetone, and oncrystallization fromithe solution substantial quantities of fiat prismsof ethyl 3,5,19-trihydroxy-A -etiocholenate were obtained, having thestructure:

19 18 CHQOH CH:

' The recrystallized crops from'three separate runs melted respectivelyat 187-189 l89191 C.,

ccpoi chloroform) Example II.--Ethyl3,5-dihydrcxy-8J9-oxidoetz'ocholanate The dry residue (brittle foam)resulting from drying the mother liquor, as described in the first twosentences of the second paragraph above, was found to contain the twotypes of substances possible from the dehydration involving the removalof the hydroxyl group originally attached to the I l-carbon, namely, (1)the product having an oxygen linkage between the 8- and 19- carbonatoms, together with (2) the product having A -unsaturation. It isbelieved that the 8,19-oxido compound results from the immediately priorformation of an 8,14-unsaturated compound, namely, ethyl3,5,l9-trihydroxy-A etiocholenate, having the structures This lattercompound is believed to be unstable and to undergo immediaterearrangement to form the oxido compound ethyl 3,5-dihydrdxy-8,l9-oxido-etiocholanate, having the structure? fomo' on, 000023 Thisoxido substance was isolated as follows: A suspension, of 350 milligramof platinum oxide in 4.5 cc. of glacial acetic acid was reduced byshaking it in an atmosphere of hydrogen. To

the resulting platinum suspension was added The catalyst was filteredoff from the hydro genation mixture and the glacial acetic acid wasremoved from, the filtrate solution by quickly bringing the solutiondown to dryness under vacuum (45-50 0.). To the resulting syrupy.residue, 5 cc. of water was added at once. After standing over night,the entire content was extracted twice with 60cc. portions each of ethylacetate and the combined extracts were washed neutral with three 3 cc.portions each of ,a 5%

solution of sodium carbonate followed 6, times with 2.5.;cc. each ofwater, and then dried with anhydrous sodium sulfate and filtered. Theresultin filtrate was evaporated to dryness, yielding-a resinous residueof 0.8278 gram. 1 0.693'7 gram of this resinous residue was dissolved in.70 cc.. ofether and fractionated by, chromatographic adsorption by,filtration within.

one hour through a millimeter diameter chum-n holding 19' grams ofBrockmann aluminumoxide, followed successively by the following eluates,each filtering-within minutes:

' CHROMATOGRAPHIO FRACTIONATION No. of Weight of Appearance 0! FractionSolvent Residue g. Residue 1 70 cc. ether (original 0.0201 resinous.

solution). 2 40 cc. ether 30 cc. 0.0912 slightly yellow, partacetone.lycrystalline. g 3 20 cc. ether 50 cc. 0.0103 yellow, crystalline.

. acetone. 4 70 cc. acetone 0. 0402 microcrystalline. 5 70 cc. acetone0.2 cc. 0. 0461 partly crystal-line methanol. resin. 6 70 cc. acetone0.3 0.0689 crystalline.

cc. methanol. 7 70 cc. acetone 0.5 0.0733 Do.

cc. methanol. 8 69 cc. acetone 1 cc. 0.0775 Do.

methanol. 0 68 cc. acetone 2 cc. 0. 0895 partly crystalline methanol.resin. 10 cc. acetone 5 cc. 0.0958 resinous.

methanol. ll 50 cc. acetone 20 cc. 0. 0407 brittle foam.

methanol. 12 cc. methanol 0,0235 amorphous.

Total 0.6761

The residue from each of the eluates was separately dissolved inacetone, and in some instances, petroleurn ether was added to inducecrystalliza- 1 tion,with the following results. i I

While no crystalline substance could be se cured from fraction 3,fractions 2 and 4 together furnished a total of 9.2 milligrams(respectively, 2.9 and 6.3 mg.) of needle-shaped crystals of ethyl 3,5dihydr0xy-8,19-oxido-etiocholanate Example III.--Ethylaim-mamaetiocholanate It was possible in the chromatographic adsorptionto isolate the compound of Example II as a product of the dehydrationbecause, during the hydrogenation that followed, it retained itsstructure. However, its isomer, formed also during the dehydration, hadits A double bond reduced by saturation during the hydrogenation.Accordingly, from fractions 6.through 10 of the chromatographicadsorption eluates, there was obtained a total of 0.1255gram ofneedle-shaped crystals identified as ethyl3,5,19-t'rihydroxyetiocholanatemelting between 178 and 182 C.

and having the structure: i

CHZOHCH} C 0 0 621515 In addition, from mother liquors from thesecrystallizationsthere was obtained a total of 0.0457 gram, which, whileof almost the same purity, had melting points between and 178 C.However, the identity of the compound in all of these fractions wasshown by mixed melting point determinations. While the total. yield thenWas 01712 gram, the yield apparently .can be somewhat increased bysubjectin the contents of the mother liquors left from the crystallizaInizthe second paragraph of Example I above,-:

it was noted thatth'e'oily residue obtained from the reaction mixtureresulting from the dehydration step wasdividedmerely'for convenienceintotwo p'artssyThe-preceding exampleswere carried out .primarilyfromworking with the'portion designated, as oily residue A. The followingEx'amdue B portions The methods in these particular examples appear.less convenient: than those "inr the preceding examples but 'yielded'anumber of. 1 chemically .pure compositions whichigserved as referencesamples. The residue B wasnot separated intoanacid fraction and" aneutral'fraction as was the resinous, oily residue 'A,'but was sub-# 7jected directly, to catalytic hydrogenation.i..

Example IVA-Ethyl 3.5-dihydo'xy 8-J9-o:cidoe etiocholanate 455milligrams of platinum oxide were suspended in 7 cc; of glacialacetieacid and lWhilB" added andthe shakingicontinued in the atmos-CHROMAT-O'GR-KPH'IC'FRKCTIONATION a- No. of 1 Weight of Appearance of1:.

solvent Residue g. 1 esidue\ r 1-; .60 icchbenzeneflorigty 0. 0006greasy;

nalsolution). 2 c%benzenc+l5'cc. 0.0020 'jresinousi" et e111 2 3 l5cc.-.benzene.-i.-45 cc. 0.0073 partly. crystalline. L

' e er; I T931112 '4. .60 cc. ether.;;....-. 0.0161 .partlyfcrystalline;i,

' .ycl1owishresin...- 5 40 rec: ether-F20 13c. 0;0127'yellowish'crystalsi acetone: 6., 20.-cc. ether-i140. cc.-. 0.0067.resinous...

acetone.- a 7.. 60cc.1acetone::=;...' 0.0102; crysta1linc:::. s 60cc.acctone-l-0.l50a...v 0.0275 Do.

methanol; i 9 60 cc.acetone-1:020cc;' 30.0.4442. V Doro:

methanol. 10 60 cc. acetone-HBO 00.. 0. 041; large crystals.

methanol." ll 60cc.acetone+0.50 cc. 0.0464 Do.

methanol. l2 58 cc. acctone+2 cc. 0.1134 crystalline.

ethan V 13 cc. acetone+5 cc. 0.0944 brittle foam.

methanol. 14 45' cc. acet0nc+l5 cc. 0.0409 resinous.

. methanol. 15 cc. methanol 0.0235 essentially crystal- V .line.-

Total 0.4878

phere of hydrogen at'room: temperature. ('23 .'C.-l

for th reezand one-halfohours,after whichthe hydrogenationmame to astandstill. Totalzhydrowere a-bs'orbed during the first hour andone-half;

Thenplatinum was' filtered.ofi and thefiltrateso-s lutionewasconcentrated: under vacuum (about;

- 52 -'.'C.): to .a:slightly turbid, colorless syruppto which: 5 .cc; ofwater .was: added immediately. 5 The next :day this, sticky material.was; taken .up... in cc. of ethyl acetate and the resulting-s01 lutionwashed withfi cc. andthen. two 2 cc. .portions each'of water.

The thus washed'ethyl acetate solution was thenseparated into aneutralfraction and an acid -fractionby taking out. the acid materialby; extraction.. successively-with10cc. and then .5

cch-ofha 5% solution ;of sodium carbonate and then :5 times with 2 cc.portions each ofwater. Theremaining ethyl acetate solution containingthecneutral fraction was-driedwith anhydrous sodium. sulfate, filteredvand-evaporated to dryness.with.:the later portion of the evaporationconductedunder vacuum. i r

Thethus. dried neutral residue-which was a white,. =largelycrystallinecake ,weighing 0.962 gram, was stirred up in someether.;which. ,dis

The "0.512 gram of this last-crystallinematerial 1 was then elaboratelypurified by chromatographieadsorpti'on, passing a solution ofit in 60"cc;- of-* benzene within a period ofthree hours through '15 grams'of-Brockmann aluminum oxide 1 in a column of-l9 mm;-{dia'meter.-Of-thefollowing eluates,"th'e-=-first four. were passed througlr' the"columnwithin an. hour :each and -the' other within about 35ininutes-eachh cholanate was obtained:

The fractions; 4 and .5. residues. from the precede ing chromatographic;fractionation were... :com-

bined anddissolvedin a verysmallvolumeofzace+ tone to which a littlepetroleum ether was added. After a few days standing, practically 3.9milligrams of stout, slightly, yellowish, prismatic crystals of ethyl3,5-dihydroxy-8,l9-oxido-etio- This product was identical with theproduct-described in Example II as well as that in Example-VIII: 1

Example V. Ethyl 3.5,1 9anhyd myretiochm lanate The fractions 3 through11 residues were sepa ratelydissolved in acetone and on a water bathconcentrated'to a small volume. Onstanding at roomtemperature; in someinstances afterthe "addition of a .little petroleum ether, long, flatprather stout prismatic crystals separated,- and afterfiltering, andconcentrating the motherliquors, additional crystal crops wereobtained'byanalogous treatment. Nine crystalline-fractions,

totaling 0.122'8 gram;- contained identical mate rial melting-"about C.The crop from franction=lo melted at l88-190---'C."and that:-from-frac--tion 11 at 188.5 C. The optical rotation of the latter crop was la-l*-|'-62.9 (20.0 mg". in 2.0

-cc.-of chloroform) i The 'fractions 12 and 13' residues were'-separa'tely recrystallized --from acetone, each yield ing one crop-of-crysta1line 'ethyl -3,5,l9-trihydroXy-etiocholanate; one weighing0.0516 gram andmelting-at18 F187 Cs "andthe other 0.0170"gram-andmelting at 184-48945 C:-

The residues from drying the mother liquors: of fractions- 8 'throughilr(0.0369 gramH-and from' fractions- 12- and '13- (0.I39'5" 'gram) were:

= -combinedandsubjected to -a similar,separate chromatographicseparation, yielding a numberaof crystalline fractions, totaling 0.0375gram 0f: I

ethyl 3,5,19-trihydroxy-etiocholanate,- melting:- above 185C! This madethe total yield's of pure samplesof this substance 02 287 gram from 7bothchromatograms; I Example V1.53,5,1'9.trihydroxy-etiochola'nicIacidj.j The"acid fractionderivedefrom the ethylacetates:

' solutionaofothe .productimf-r the .ahydrogenationel little acetone.

described in Example IV (second paragraph, first sentence; contained inthe combined two sodium carbonate extracts and five water washings whichfollowed them), as so combined, was made acid to Congo paper by theaddition of 2.5 cc. of concentrated hydrochloric acid while cooling bythe addition of ice. This caused the formation of a cheesy precipitatewhich was then thoroughly extracted with separate portions ofethylacetate, first 75 00., then 50 cc., and then three of 40 cc. each.These ethylacetate extracts were combined and washed six times with 2cc. each of water, and then dried with anhydrous sodium sulfate andfiltered. The filtrate solution, was evaporated to dryness yielding0.1881 gram of a colorless resin as the acid fraction.

This acid fraction was then dissolved in .a On standing overnight, thereseparated 11.4 milligrams of crystalline 3,5,19- trihydroxyetiocholanicacid melting at 2:10-2 i7 C. (to a light brown liquid, followed byeffervescence), and having the structure:

CHQOH CH3 O OH From the mother liquor there separated a secondcrystalline crop, 26.0 milligrams, melting between 210-230 C. (to ayellow liquid, followed by effervescence) 1 The dehydration described inExample I (column- 3) did not go to completion because it yielded acertain amount of ethyl 3,5,14,19-tetrahydroxy-l4-iso-etiocholanate aspointed out in column 17. Accordingly, a second dehydration was carriedout under slightly more vigorous conditions.

1.2716 grams of recrystallized3,5,14,19-tetrahydroxy-14-iso-etiocholanic acid was dissolved in 130 cc.of a one-tenth normal solution of hydrogen chloride in absolute alcohol,and refluxed (kept at a temperature of 87 C.) over a paraffin bath forone hour. It was then distilled at atmospheric'pressure at a slow ratewith the bath temperature at 92-95 C. for 90 minutes, and finally at amore rapid rate, with the bath temperature raised to 100 C. for 45minutes. To the residue, about 40 cc. of a yellowish solution, there wasadded 30 cc. of water, and the distillation then continued under vacuum(45-50 C.) until there appeared a distinct turbidity. This was broughtinto solution 'by briefly heating on a water bath. The solution was thenallowed to stand overnight at room temperature. There 10 producing inthe residue an oily precipitate. The latter dissolved by the addition of100 cc. of ether, and the resulting lower aqueous phase was separatedand extracted 5 times with cc. portions each of ether, which etherwashes were combined with the original ether phase. The combined ethersolution was washed five times with 3 cc. portions each of water andthen separated into a neutralfraction and an. acid fraction .byextracting this ether solution successively with 10 cc. and then 5 cc.of a 5% solution of sodium carbonate followed by 7 washes with 3 cc.each of water. The two sodium carbonate extractions and the seven waterwashes were combined and contained theacid fraction extracted from theresinous, oily material, and their further treatment is described inExample X.

The remaining ether solution (now freed of the acid fraction) containingthe neutral fraction, was dried with sodium sulfate which was thenfiltered oil, and the solution evaporated to dryness yielding 1,085grams of a colorless, brittle foam as the neutral residue.

Petroleum ether was then added very gradually (as described in column4), to a solution of this neutral residue in 4 cc. of ether, and thereformed only a resinous precipitate. This, together with the supernatantsolution, was. then evaporated to dryness and subjected to catalytichydrogenation thus:

450 milligrams of platinum oxide were suspended in 7 cc. of glacialacetic acid and, while being shaken, were reduced in an atmosphere ofhydrogen. Then a solution of 1.080 grams of the dried neutral residue in12 cc. of glacial acetic acid was added and the shaking continued in anatmosphere of hydrogen at room temperature (26 C.) for three andone-half hours. Total hydrogen absorption was 68.7 cc. The platinum wasfiltered ofi and the filtrate solution immedi ately evaporated undervacuum (45-50 C.) to dryness. To the syrupy residue, 5 cc. of water wasadded at once. The next day the wax-like,

semi-crystalline material was dissolved in cc.

; of ethylacetate, and the water phase was separated from theethylacetate solution phase which separated some resinous material andalso yellowresin from the crystals by aqueous alcohol (1:l).

The dry weight of the yellow needle crystals was 45.8 milligrams,melting at 91-93" C.

Example VIA-Ethyl 3,5,19-trz'hydroxyetiocholanate Following theimmediately preceding separation of the crystals from the mother liquorcontaining the resinous material, the alcohol was completely removed bydistillation under vacuum,

latter was made neutral by washing it successively with 5 cc. and then 2cc. of a 5% solution 01' sodium carbonate followed by five washings of 2cc. each of water. After drying with anhydrous sodium sulfate andfiltering off the latter, the ethylacetate was removed by heating undervacuum continued until the residue was brought completely to dryness,yielding 1.0404 grams of resinous residue. This residue was thendissolved in a small volume of acetone and after seeding with anauthentic sample of ethyl 3,5,19-trihydroxyetiocholanate, separation ofstout, prismatic crystals began immediately. The following day thecrystals were filtered off and dried, weighing 0.3474 gram melting atabout 170-175 0., and identified as moderately pure ethyl 3,5,19-trihydroxy-etiocholanate, giving a mixed melting point of 177 C. with apure authentic sample of the substance.

Example VIIL-Ethyl 3,5-dihydroary-8,19-o:nidoetiocholanate From the lastfiltrate, 0.0368 gram of definitely impure crystalline material, meltingat -158 0., was obtained, the mother liquor from which, on being broughtcompletely to dryness, gave 0.5740 gram of resinous residue. The residuewas dissolved in 60 cc. of ether and subjected to chromatographicadsorptionby filtration within 11 80 minutes through 16 grams ofBrockmann aluminum oxide in a mm. diameter column. The following eluateswere obtained after passing through within -40 minutes. each.

GHROMATOGRAPHIO FRACTIONATION No.01 Weight of Appearance of z- SolventResidue g. Residue 1.. '60 cc. ether (original 0.0603 resinous.

solutio 2 .40 cc. ether-l-QO 'cc. 0.1382 partly crystalline acetone.resin.

3 .20 cc. ether+-cc. 0.0251. crystalline.

, acetone.

4 60 cc. acetone 0.0368 Do. 5.. -60 cclacet0ne+0.l5cc. 0.0255 partlycrystalline methanol. 7 g resin.

6.. 60 cc.acetone+0.20 cc. 0.0097 crystalline.

methanol. H

7 60 cc. acetone+0.30cc. 0.0065 resinous.

I 'methan I 8. ."60cc.acctone+0.40cc.; 0:011'7, partly crystal-linemethanol. resin.

9 60 cc.acetone+0.50 cc. 0.0288 crystalline. I

methanol.

10.- 59 cc. acetone+l cc. 0.0461 Do.

methanol.

11 58 cc. acetone-+2 00. 050640 ioamyglass.

methanol. v

l2 .1 cc. acetone+5 cc. 0.0647 Do.

' methanol. 7

13 45 cc. acetone-H5 cc. 0.0293 Do.

methanol.

l4 .4 cc. methanol 0.0153 semi-crystalline.

Total 0. 5680 milligrams. When tetranitromethane was added to a sampledissolved in chloroform, no yellow color was observed.

Emample IX.-Ethyl 3,5,19-trihydroazye etiocholanate When the fractions 9and 10 residues were recrystallized from acetone, a'total of only 40.8milligrams of transparent rod-shaped crystals, melting points between C.and C., were obtained, of moderately pure ethyl3,5,l0-trihydroxyetiocholanate, as established by the determination ofmixed melting point with an -au thentic sample.

While the dehydration of the tetrahydroxy starting material was carriedoutin Examples 1 and .VI by the use ofabsolute ethyl alcohol, the samedehydration takes place when there is used instead any othersuitable'absolute alcohol, such as any of the other absolute loweraliphatic alcohols as methyl, propyl, isopropyl, or butyl alcohols, andthe like. By such substitution there is obtained in the steps after thedehydration, instead of an ethyl ester of the unsaturated tri hydroxyacid, the 8,19-oxidodihydroxy acid and the saturated trihydroxy acid,the resulting methyl 3,5,19-trihydroxy-A etiocholenate and methyl3,5-dihydroxy-8,19-oxidoand 3,5,19 trihydroxy etiocholanates; propyl3,5,19 trihydroxy A etiocholenate, and propyl3,5-dihydroxy-8,19-oxidoand 3,5,19-trihydroxy-etiocholenates, and thecorresponding isopropyl ester of each of this one unsaturated acid andof the two saturated acids; and butyl 3,5,19-trihydroxyn -etiocholenate,and butyl 3,5-dihydroxy-8,19- oXido-, and3,5,19-trihydroxy-etiocholanates, and the like other lower alkyl estersof the corresponding three acids.

1'2 7 Example X.3,5,19-trihydromy-A -etioch0Zenic acid The combinedsodium carbonate extractions and water washings containing the acidfraction" extracted from the resinous, oily material, as described inthe first'paragraph of Example VII,

COOH. On

Example XI .3,5,1 9-trihydroa:y-etiocholanic acid (a) 50 milligrams ofplatinum oxide were susended in 3 cc. of glacial acetic acid, andreduced with shaking in an atmosphere ofhydrogen. Then a solution ofthis 0.1186 gram of acid residue in 4 cc. of glacial acetic acid wasadded and the shaking in an atmosphere of hydrogen continued for threehours; total hydrogen absorption 8.9 cc. at 26 C. The platinum was thenfiltered off and the solution filtrate brought to dryness under vacuum.To the resulting resinous residue was added 3 cc. of water, and bykneading. it, on the following day, several times with 0.5 cc. portionseach of water, it finally became crumbly and filterable. Afterfiltration and drying, its

-. weight was 0.094 gram. This material recrystalliquor, 5.8 milligramsof crystalline material,

melting at 245-252 C., was obtained.

(b) While in section (a) of this example the. product of the example wasobtained by using as starting material the product of Example X andconsequently hydrogenating the unsaturated corresponding A -etiocholenicacid contained in that acid residue starting material, the samesaturated acid of this Example XI was obtained by saponifi cation of thecorresponding alkyl 3,5,19-trihydroxy-etiocholanate, thus:

0.17 gram of potassium hydroxide dissolved in 1.0 cc. of methanol wasadded to a solution of 86.8 milligrams of purest ethyl 3,5,19-trihydroxyetiocholanate (as derived from Example III, first paragraph) in 2.5 cc.of methanol. Afterrefiuxing the mixture on a water bath for two hours, 2cc. of water was added, and the solution brought almost to dryness undervacuum, and the residue 13 taken up in -6 cc. of water. The suspendedcrystalline material was extracted by shaking with cc. of ether, and theether phase washed three times with 1 cc. portions each of water. Afterdrying with anhydrous sodium sulfate and then filtering it off, theether solution filtrate was brought to dryness under vacuum, yielding29.5 milligrams of crystalline neutral residue.

The aqueous washings were combined with the alkaline solution phase andthe whole made acid ,dissolve all of the suspended white material.

Then the aqueous phase was extracted once more with lo cc. of ethylacetate. All of these extracts were combined and washed once with 2 cc.and

'4 times with 1 cc. portions each of water, and

after; drying with anhydrous sodium sulfate and then filtering it off,the filtrate solution was evaporated to dryness under vacuum. Thiscrystalline acid residue, dried in a vacuum desiccator over solid KOH,and weighting 53.8 milligrams, was dissolved in the required amount(10-11 cc.) of acetone. On concentrating the solution over a water bathto a smaller volume (about 2 cc), suddenly crystallization of thesubstance in bunches of flat, spear-shaped crystals set in, and thecrystallizing solution was left standing at room temperature for a fewhours. After filtering off the crystals and drying them, they weighed28.2 milligrams, melting at 259-260 C. By coricentrating the motherliquor additional crops of rather pure product were obtained, one of15.1 milligrams melting at 256.5-258 C., and another of 1.8 milligramsmelting at 254-256" C. All three crops melted to alight brown liquidwith subsequent effervescence. Optical rotation, determined on the firstcrop, was +69.5 (8.0 mg. in 2.0 cc. of acetone).

Example XII-Methyl 3,5,19-trihydroxy etz'ocholcmate (a) A solution inether of 29.5 milligrams of the neutral crystalline residue (from thepenultimate paragraph of Example XI) was concentrated on a water bathuntil crystallization started. The next day 17.! milligrams of stout,ruler-shaped crystals were filtered off, melting at 208211 C., obviouslyrepresenting a mixture of,

the ethyl and methyl esters of 3,5,19-trihydroxyetiocholanic acid.

By concentrating the filtrate on a water bath,

4.8 milligrams of additional crystalline material,

melting at 2225-224" C. was recovered. This was pure methyl3,5,19-trihydroxy-etiocholanate. It caused no depression of meltingpoint when mixed with an authentic sample of the compound. On bringingdown to dryness the mother liquor from this last crop, 6.1 milligrams ofa crystalline,

. methanol, these same alkyl and other esters can also be obtained bydirect esterification of the 14 free acid with a suitable diazo reagent,for example, a diazo-alkane or aromatic diazonium salt. The presentsection of this example illustrates this alternate method:

To a solution of 26.6 milligrams of 3,5,l9-trihydroxy-etiocholanic acidin 5 cc. of acetone, there was added at 0 C. a slight excess of anethereal solution of diazo-methane. After standing at room temperaturefor twenty minutes, the excess diazomethane was removed over a waterbath and then the resulting colorless solutionwas evaporated to drynessunder vacuum. The crystalline residue was dissolved in 15 ccoof etherand this solution made neutral by washing it successively with 1 cc. ofnormal hydrochloric acid, 1 cc. of water, 1 cc. of a 5% solution ofsodium carbonate, and 3 times with 1 cc. portions each of water. Thesolution was then dried with anhydrous sodium sulfate and, afterfiltration, was evaporated to a small volume on a water bath.

- Crystallization started spontaneously in characteristic hexagonalplatelets. Afterstanding for sometime at room temperature, the methyl3,5,19-

trihydroxy-etiocholanate was filtered off and dried, weight 15.2milligrams, melting at 220 222.5 0.; optical rotation [a] +61.3 (8.0 mg.in 2.0 cc. of chloroform). After concentrating the mother liquor, asecond crop of 3.8 milligrams of similar looking crystals, melting at218-220.5 C., was obtained.

Other similar esters of 3,5,19 trihydroxy etiocholanic acid are obtainedby replacingthe diazo-methane of Example XII, (b) by the correspondingdiazo reagent, RN2, in which Ris the hydrocarbonradical replacing themethyl group in the methyl ester of the example, such as reacting saidacid starting material under 1 reaction conditions asin Example XII .(b)with the required corresponding diazoalkanez or with the desiredcorresponding diazo (lower)- alkane as diazoethane, diazopropane,diazobutane, and the like, or by reacting the3,5,19-trihydroxy-etiocholanic acid with the desired corresponding aryldiazonium salt to introduce the desired aryl group when R (in thegeneral formula of claim 1) is aryl, for example, with a phenyldiazonium halide as the chloride, and

. the like, and obtaining phenyl 3,5,19-trihydroxyetiocholanate, and thelike other aryl esters of thisflacid. The corresponding esters, bothwhen R is an alkyl as well as an aryl hydrocarbon, can be made also bythe procedure of esterifying the l'l-carboxyl group with an alcohol.This procedure is used. when instead of the methyl group as in the esterof Example XII, there is desired the corresponding ester having anaralkyl group as benzyl, for example, obtained by esterification of theacid with benzyl alcohol, yielding benzyl 3,5,19-trihydroxy-5hydroxy-etiocholanate.

Example XIII.--Ethyl 3,19 diacetoxy 5 hydroary-etiocholanate To asolution of 108 milligrams of pure .ethyl3,5,19-trihydroxy-etiocholanate in 0.4 cc of pyridine there was added0.4 cc. acetic ,anhydride, and the mixture allowed to standat roomtemperature for 22 hours. It was then concentrated under vacuum (65 C.)to a viscous colorless oil which was then dissolved in 25 cc. of ether,neutralized, by shaking it successively twice with 2 co. portions eachofnormal hydrochloric acid, twice with 2 cc. each of a 5% solution ofsodium carbonate and 5 times with 1 cc.

each of water, and then dried with anhydrous '30. minutes.

2,584goo1 sodium sulfate. 1 After filtering oil" the latter,

the-solution"filtrate was evaporated to dryness leaving a colorlessresin -(weight 12'7.5 mg.) which partly crystallized on standingovernight in a vacuum desiccator.

completely into'solution, and then after stand- -ing ashort time, somewhite crystals of ethyl 3l9 'diacetoxy 5 hydroxy etiocholanate separatedout. After filtering these off i and drying them'-(weight first :crop39.0 mg.) they. melted at--108-l09.5 0., and showed opticalrotation of.[a] +60;3 (20.0 mg. in. 2.0 'cc. of

"-chloroiorm) Uponconcentrating' the filtrate to a smallervolume,-=filtering 'off the additional crystals-and -::similar1yconcentrating -the subsequent filtrat'es;additional'crops were obtained,the first :of 31.3 milligramsmelting at 107-108 C., the

secondfof 22.4 milligrams melting at 103-105. 0.,

andaxthe last of 12.4.- milligrams melting! at Ifrin the procedurerofthis Example: XIII, the

: acetic anhydride is replaced by the corresponding anhydride of another.aliphatic acid or aro- ;matic or aryl-aliphatic acid, there is obtained.the corresponding ethyl 3,19-dialkoyloxy (or ,saroyloxy or:-ar'alkoyloxy) --hydroxy-etiocholanate, for example, ethyl3,19-dibenzoyloxy-5- hydroxy-etiocholanate.

Likewise, if instead of starting with the ethyl3,5,19-trihydroxy-etiocholanate, there was used instead in Example XIIIany other l7-alkyl (or EammpleXIV.--3,5-dihyclroxy-8,19-oridoctiocholanic acid 'i 'To a solutionof34.9'milligramsof ethyl 3,5-

" dihydro'xy-8,19-oxido-etiocholanate dissolved in 11cc. of .methanolwas added '70 milligrams of ypotassium hydroxide dissolved in 1 cc. ofmethanol. The mixture was refluxed for two hours and then kept at roomtemperature for about Thereafter 3 cc. of water 'was added and thesolution'concentrated under vac- ?uum to about 100. volume. After adding3 cc. of waten-the resulting turbid solution was ex- "tracted four timeswith 5 cc. portions each of 'ether, and the ether phases were. combined.The combined ether phases were then washed three times with 1.5 cc. eachof water, the separate water'layers drawn off; and the washed ethersolution dried with anhydrous-sodium sulfate'and then filteredandthe-solution filtrate evaporated to dryness, leaving 4.1 milligrams ofcrystalline neutral residue.

separated promptly a white, apparently crystal- -line precipitate whichwas then extracted '2' times "with 5 cc. portions each of ethyl acetate.The

-;"ethyl-acetate extracts were'combined, Washed 5 times with 1 cc. eachof water, dried with anhydrous sodium sulfate and filtered. The solutionfiltratewas evaporated completely to dry- -mess and then kept o-vernight in a vacuum desiccator over potassiumhydroxide, yielding 27.6

7 After adding alittle ether- .-'-to the resin-crystal mixture,'thematerial-went milligrams of crystalline acid residue whichwas -aryl, or-aralkyl) ester, and theprocedure cara .dryness to a white residue.

then dissolved in 1000. of acetone and the 50141- tion-concentrated on aWater bath to about 3 cc. After standing atroom temperature "for a fewhours, white, scaly microcrystals of3-,5-dihydroxy-8,19-oxido-eti0cho1anic acid were -filtered off, havingthe structure:

Li y.

weight 6.6 milligrams, melting at 2ac-2 ss c, "optical rotation [el+77.3 (3.6 mg. in 2.0 cc.

of acetone) By concentrating them'other liquor, filtering offthe'crystals obtained, and'repeating with the subsequent filtrationmother liquor s,fadditional crops of fairly pure "crystalsweregobtained, the second crop weighing" fifi-"r'n'illigrams and meltingat 2855-2875 C.; the third "of"2.9 milligrams melting at 281-283" 0.,and the-fourth of 0.6 milligram melting at 283-286? C. The final motherliquor. was evaporated "under vacuum to dryness, leaving 8.9- milligramsof crystalline residue.

Example X V.-M ethyl 3,5-dihl/d703211-8t19:0.73id0- .etiocholanate n;

.(a) 8.9 milligrams of. ,crysta11ine a5-d1hydroxy- 8,19 -,oxidoetiocholanic acid 1. (Example fziIV)'g.was dissolved in 6.00. of acetoneand-concentrated 'over a waterbath .to. about 4 cc. ,f'To

the solution at '0v C. was. added an. excess; of

ethereal solution-of diazomethane and. themixture allowied tostandin-the coldfor-aboutilO minutes and then at room temperature about. 25minutes, and then evaporated; under vacuurn to sulfate and after.filtering oil the latter, was n evaporated to dryness,yielding-9.8;milligramsof White crystalline residue. This residue wasthen treated with some acetone and after filteringjfoff the whitecrystalline material, the filtrateyielded 4.9 milligrams of microscopic,flat shortlneedle crystals of the methyl 3,5-dihydroxy8gl9:oxidO-etiocholanate, melting at 270-274 C("(t'oa brown liquid).

residue was obtained by evaporating the filtrate from the needlecrystals,.underyacuum'to dryness.

(b) The 8.9 milligram crystalline residue. of

3,5 -dihydroxy 8,19 oxido-etiocholanic acidiobligrams of resinousresidue was obtained.

cholanic acid are obtained byreplacing-the di- This residue. was

3.3 milligrams, of "white crystalline azo-methane by the requiredcorresponding diazo-alkane, or corresponding aryl diazonium salt, andfollowing the procedure of this example, or by the procedure ofesterifying the carboxyl group with an aralkyl alcohol, as explained inthe paragraph preceding Example XIII.

By reacting 3,5,19- trihydroxy etiocholanic acid, or either thecorresponding A -etiocholenic or -8,l9-oxido-etiocholanic acids, or anyalkyl, aryl, or aralkyl ester of either of these three acids, with anesterifying agent, for example. an

acid anhydride as acetic anhydride or phenylacetic anhydride, or anacid'halid'e as benzoyl chloride, or.an acid as glacial acetic acid orphenylsulfonic acid, under mild esterification conditions there resultsthe corresponding derivatives of each of the three acids or of any oftheir indicated 17-carboxylic esters, in which the hydroxyl groupsattached to both the 3- and 19- carbon atoms are converted to alkoyloxy,aroyloxy, aralkoyloxy, or arylsulfonyloxy groups, for example, 3,19 di(phenylsulfonyloxy) hydroxy-etiocholanic (or A -etiocholenic) acid and 3phenylsulfonyloxy 5 hydroxy 8,19 oxidoeti'ocholanic acid, or thecorresponding alkyl (aryl or aralkyl) 3,19-di-(phenylsulfonyloxy)-5-hydroxy-etiocholanic (or n -etiocholenic) acid and3-phenylsu1fonyloxy-5-hydroxy-8,19-oxidoetioeholanic acid.

Example XVI.-Laetone of empirical formula CzoHzsOr This substance provedto be essentially insoluble in a 5% solution of sodium carbonate. Ontreating a chloroform solution of it with tetranitromethane, a distinctyellow color, indicative of its unsaturated character, formed. Itsrecrystallization was difficult.

Example X VII .-Ethyl 3,5,1 4,1 Q-tetrahydrozry-I 4- iso-etiocholanateThe hard white crystals obtained from the neutral portion extracted fromthe oily residue A following the original dehydration step (as dewasrecrystallized from a small volume of acetone to which an equal amountof petroleum ether was added, About 11 milligrams. separated ascrystalline scales, melting at 186-189 C., and showing no depression ofmelting point when mixed with an authentic sampleof ethyl 3,5,1e,l9-tetrahydroxy-14-iso-etiocholanate. This compound illustrates anotherexample of the alkyl esters of 3,5,14,19tetrahydroxy-l4-iso-etiocholanic acid, which alkyl esters of this acidare referred to in the last paragraph of Example 5 of application SerialNo. 666,191, now Patent 2,518,672.

All melting points were determined with the Fisher-Johns melting pointapparatus. The readings were suificiently near the true melting pointsso that no corrections have been made.

The products of the invention are useful as intermediates in thepreparation of other compounds, and some of them have certaintherapeutic effectiveness.

It will be understood, with reference to the various compoundsillustrated and described in this specification and its claims, that Ido not intend that the invention of any of the compounds described orclaimed shall be limited to any particular stereochemical configurationabout any carbon atom, and, in particular, about carbon atoms 3,5,10,14and 17.

The 3,5,14,l9-tetrahydroxy-le-iso-etiocholanic acid starting materialfor the products described in this specification is prepared byoxidizing, for example, 5.0 grams of strophanthidol-3,l9-diacetatedissolved in 294. cc..of acetone with 4.84 grams of potassiumpermanganate, distilling off the acetone under vacuum, diluting theresidue with water to produce a sludge, acidifying the sludgy mixture toCongo paper with 10% suliuric acid,'extracting the sludge with ether,extracting an acidconstituent from the ether extract by treating thelatterwith 5% solution of sodium carbonate, making the sodium carbonateextraction acid to Congo paper using 10% sulfuric acid, causing astickyprecipitate to appear, extracting the precipitate mixture withether, washing the ether extract with water and drying it with anhydroussodium sulfate, filtering oif the latter, and bringing the solutionfiltrate completely to dryness under vacuum, yielding 3,5,14,19-tetrahydroxy l4 iso etiocholanic acid, 3,19- diacetate; then hydrolyzingthis, diacetate in methanol, for example, 3 grams in 15 cc. of methanol,and adding a solution of 4.5 grams of potassium hydroxide in 60 cc. ofmethanol, refluxing on a water bath, adding cc. of water, concentratinunder vacuum to about 65 cc., cooling the solution by immersing itscontainer in an ice bath, and making the solution acid to Congo paper,using concentrated hydrochloric acid, extracting with ethyl acetate theresulting mixture, washing the extract with water, and then drying itwith anhydrous sodium sulfate, filtering on and concentrating thefiltrate under vacuum to about 16-45 cc., permitting the separation ofmicrocrystalline 3,5,14,19-tetrahydroxy-14-iso-etiocholanic acid tocontinue overnight.

This application is in part a continuation of my copending applicationSerial No. 713,925, now abandoned, which in turn is in part acontinuation of application Serial No. 666,191, now Patent 2,518,672.

While the invention has been illustrated by certain specific embodimentsof it, it is understood 7 that various substitutions and modificationsmay CHI- CH1 0008 Rio CHQORI CH: 000R v 20 3. The compound:

VOHIOH 0000,133-

4. The compound:

- onion CH: 0000,11s

5. In the process of preparing a 3,5-dihydroxy-IO-hydroxymethane-l'7-carboxy-estrane, the step of dehydrating3,5,14,19-tetrahydroxy etiocholanic acid by the use of acidulatedalcohol thereby removing the hydroxyl group from the l4-position forminga compound selected from the class consisting of those in which (a) thecarbon atoms in the 10- and 8-positions are joined by a methylenoxylinkage and the carbon atoms in the 14- and 15-positions are joined by asingle bond, and (b) the carbon atoms in the 8- and l-positions arejoined by a single bond and the carbon atoms in the 14- andIii-positions are joined by a double bond; and simultaneouslyesterifying the 17-carboxyl group.

6. The process as claimed in claim 5 wherein the dehydration isperformed under anhydrous conditions.

7. The process as claimed in claim 5 wherein the alcohol is acidulatedwith a mineral acid.

MAXIMILIAN R. EHRENSTEIN.

No references cited.

1. A COMPOUND SELECTED FROM THE GROUP CONSISTING OF: